Development of microsatellite markers in Cannabis Sativa for fingerprinting and genetic relatedness analyses

Microsatellite markers were developed for Cannabis sativa L. (marijuana) to estimate the level of polymorphism, usefulness for DNA typing (genotype identification), and to measure the genetic relationships between the different plants. Twelve different oligonucleotide probes were used to screen an enriched microsatellite library of Cannabis sativa in which 49% of the clones contained microsatellite sequences. Characterization of microsatellite loci in Cannabis revealed that GA/CT was the most abundant class of isolated microsatellites representing 50% overall. Eleven polymorphic SSR markers were developed, derived from dinucleotide motifs and eight from trinucleotide motifs. A total of 52 alleles were detected averaging 4.7 alleles/locus. The expected heterozygosity of the eleven loci ranged between 0.368 and 0.710 and the common probability of identical genotypes was 1.8 x 107. The loci identified 27 unique profiles of the 41 Cannabis samples. The eleven microsatellite markers developed in this study were found to be useful for DNA fingerprinting and for assessing genetic relationships in Cannabis.

Twelve new polymorphic microsatellite markers from the loggerhead sea turtle (Caretta caretta) and cross-species amplification on other marine turtle species

[EN] We describe 12 new polymorphic dinucleotide microsatellite loci and multiplex Polymerase Chain Reaction conditions from the loggerhead sea turtle Caretta caretta. Levels of polymorphism were assessed in 50 individuals from the nesting population of the Cape Verde Islands.

Characterization, development and multiplexing of microsatellite markers in three commercially exploited reef fish and their application for stock identification

Thirty-four microsatellite loci were isolated from three reef fish species; golden snapper Lutjanus johnii, blackspotted croaker Protonibea diacanthus and grass emperor Lethrinus laticaudis using a next generation sequencing approach. Both IonTorrent single reads and Illumina MiSeq paired-end reads were used, with the latter demonstrating a higher quality of reads than the IonTorrent. From the 1–1.5 million raw reads per species, we successfully obtained 10–13 polymorphic loci for each species, which satisfied stringent design criteria. We developed multiplex panels for the amplification of the golden snapper and the blackspotted croaker loci, as well as post-amplification pooling panels for the grass emperor loci. The microsatellites characterized in this work were tested across three locations of northern Australia. The microsatellites we developed can detect population differentiation across northern Australia and may be used for genetic structure studies and stock identification.

Highly polymorphic microsatellite markers for the Mediterranean endemic fan mussel Pinna nobilis

Pinna nobilis is an endemic bivalve of the Mediterranean Sea whose populations have decreased in the last decades due to human pressure; as a consequence, it was declared a protected species in 1992. Despite its conservation status, few genetic studies using mitochondrial markers have been published. We report on the isolation and development of 10 microsatellite loci for the fan mussel, Pinna nobilis. All loci (2 di-nucleotide, 5 tri-nucleotide, 2 tetra-nucleotide and 1 penta-nucleotide) are characterized by high levels of polymorphism in 76 individuals tested from two populations in the Balearic Islands (Spain, Western Mediterranean Sea). The number of alleles ranged from 4 to 24 and expected heterozygosity ranged from 0.4269 to 0.9400. These microsatellites could be very useful for the assessment of the genetic diversity and connectivity patterns of P. nobilis and the establishment of new conservation strategies.

Characterization of 10 new tetranucleotide microsatellite markers for the European eagle owl, Bubo bubo: Useful tools for conservation strategies

Bubo bubo is the largest owl in the world, showing a wide geographical distribution throughout the Palaearctic region. It underwent a demographic decline in many European countries during the last century and was considered “vulnerable” (Annex II of the CITES). Nowadays, it is classified as “Least Concern” according to IUCN. Despite its ecological importance and conservation status, few polymorphic molecular markers are available to study its diversity and population genetics. We report on the isolation and development of 10 new microsatellites for the Eagle owl, B. bubo. All loci (10 tetra-nucleotide) are characterized by high polymorphism levels. Number of alleles ranged from 5 to 13 and expected heterozygosity varied from 0.733 to 0.840. These microsatellites would be very useful to assess the genetic diversity, connectivity patterns and parentage of B. bubo. This information will allow to establish new conservation strategies and improve the management of the species.

Transcriptome analysis of Euterpe edulis and identification of microsatellite markers.

2016

Genetic diversity of mango accessions (Mangifera indica) using new microsatellite markers and morphological descriptors.

Genetic diversity estimates based on morphological and molecular data can provide different information on the relationship between cultivars of a species. This study aimed to develop new microsatellite markers as additional tools in genetic studies on mangoes (Mangifera indica L.), and to analyze the genetic variability of 20 mango cultivars based on morphological descriptors and microsatellite markers. We aimed to better understand the cultivars enhanced breeding histories and to support crossbreeding planning. Positive clones were selected from a DNA library enriched for microsatellite regions for sequencing and primer design. Four plants of each of the 20 accessions were used for observations, based on 48 morphological descriptors. Twenty accessions were analyzed using 27 microsatellite markers, of which 16 were developed during this study. The clusters, based on the morphological descriptors by Ward - MLM strategy and the microsatellite markers, suggested that Brazilian mango cultivars have extensive genetic diversity and are related to cultivars with different provenances, demonstrating their different enhanced breeding histories.

Isolation of microsatellite loci in the Capricorn silvereye, Zosterops lateralis chlorocephalus (Aves: Zosteropidae)

The Capricorn silvereye (Zosterops lateralis chlorocephalus) is ideally suited to investigating the genetic basis of body size evolution. We have isolated and characterized a set of microsatellite markers for this species. Seven out of 11 loci were polymorphic. The number of alleles detected ranged from two to five and observed heterozygosities between 0.12 and 0.67. One locus, ZL49, was found to be sex-linked. This moderate level of diversity is consistent with that expected in an isolated, island population.

Construction of microsatellite linkage maps for Corymbia

The genus Corymbia is closely related to the genus Eucalyptus, and like Eucalyptus contains tree species that are important for sub-tropical forestry. Corymbia's close relationship with Eucalyptus suggests genetic studies in Corymbia should benefit from transfer of genetic information from its more intensively studied relatives. Here we report a genetic map for Corymbia spp. based on microsatellite markers identified de novo in Corymbia sp or transferred from Eucalyptus. A framework consensus map was generated from an outbred F 2 population (n = 90) created by crossing two unrelated Corymbia torelliana x C. citriodora subsp. variegata F1 trees. The map had a total length of 367 cM (Kosambi) and was composed of 46 microsatellite markers distributed across 13 linkage groups (LOD 3). A high proportion of Eucalyptus microsatellites (90%) transferred to Corymbia. Comparative analysis between the Corymbia map and a published Eucalyptus map identified eight homeologous linkage groups in Corymbia with 13 markers mapping on one or both maps. Further comparative analysis was limited by low power to detect linkage due to low genome coverage in Corymbia, however, there was no convincing evidence for chromosomal structural differences because instances of non-synteny were associated with large distances on the Eucalyptus map. Segregation distortion was primarily restricted to a single linkage group and due to a deficit of hybrid genotypes, suggesting that hybrid inviability was one factor shaping the genetic composition of the F2 population in this inter-subgeneric hybrid. The conservation of microsatellite loci and synteny between Corymbia and Eucalyptus suggests there will be substantial value in exchanging information between the two groups.

Grey Mackerel (Scomberomorus semifasciatus) microsatellite genotypes (nine loci) representing twelve regions along Australia's northern coastline from the eastern coast of Queensland to the western coast of Western Australia.

Scomberomorus semifasciatus is an Australian endemic found in tropical, coastal waters from eastern to western Australia. Commercial and recreational exploitation is common and regulated by state-based authorities. This study used mitochondrial DNA sequence and microsatellite markers to elucidate the population structure of Scomberomorus semifasciatus collected from twelve, equidistant sampling locations. Samples (n=544) were genotyped with nine microsatellite loci, and 353 were sequenced for d-loop (384 bp) and ATP (800bp) mitochondrial DNA gene regions. Combined interpretation of microsatellite and mtDNA data identified four genetic stocks of S. semifasciatus: Western Australia, northwest coast of the Northern Territory, Gulf of Carpentaria and the east coast of Queensland. Connectivity among stocks across northern Australia from the Northern Territory to the east coast of Queensland was high, but in contrast, there was a clear genetic break between populations in Western Australia compared to the rest of northern Australia. This indicates a restriction to gene flow possibly associated with suboptimal habitat along the Kimberley coast (northwestern Australia). The appropriate scale of management for this species corresponds to the jurisdictions of the three Australian states, except that the Gulf of Carpentaria stock should be co-managed by authorities in Queensland and Northern Territory.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Polymorphic microsatellite loci for primitively eusocial wasp Ropalidia marginata

We report here development and characterization of 48 novel microsatellite markers for Ropalidia marginata, a tropical, primitively eusocial polistine wasp from peninsular India. Thirty-two microsatellites showed polymorphism in a wild population of R. marginata (N = 38) collected from Bangalore, India. These markers will facilitate answering some interesting questions in ecology and evolutionary biology of this wasp, such as population structure, serial polygyny, intra-colony genetic relatedness and the pattern of queen succession.

White shark (Carcharodon carcharias) microsatellite genotypes (six loci) from regions along Australia's southern and eastern coastline.

Despite international protection of white sharks (Carcharodon carcharias), important conservation parameters such as abundance, population structure and genetic diversity are largely unknown. The tissue of 97 predominately juvenile white sharks sampled from spatially distant eastern and southwestern Australian coastlines was sequenced for the mitochondrial DNA (mtDNA) control region and genotyped with six nuclear-encoded microsatellite loci. MtDNA population structure was found between the eastern and southwestern coasts (FST = 0.142, p < 0.001), implying female natal philopatry. This concords with recent satellite and acoustic tracking findings which suggest the sustained presence of discrete east coast nursery areas. Furthermore, population subdivision was found between the same regions with biparentally inherited microsatellite markers (FST = 0.009, p <0.05), suggesting that males may also exhibit some degree of reproductive philopatry. Five sharks captured along the east coast had mtDNA haplotypes that resembled western Indian Ocean sharks more closely than Australian/New Zealand sharks, suggesting that transoceanic dispersal or migration resulting in breeding may occur sporadically. Our most robust estimate of contemporary genetic effective population size was low and below the threshold at which adaptive potential may be lost. For a variety of reasons, these contemporary estimates were at least one, possibly two orders of magnitude below our historical effective size estimates. Further population decline could expose these genetically isolated populations to detrimental genetic effects. Regional Australian white shark conservation management units should be implemented until genetic population structure, size and diversity can be investigated in more detail. Reference: Blower, D. C., Pandolfi, J. M., Gomez-Cabrera, M. del C., Bruce, B. D. & Ovenden, J. R. (In press - April 2012). Population genetics of Australian white sharks reveals fine-scale spatial structure, transoceanic dispersal events and low effective population sizes. Marine Ecology Progress Series.